The general objective of the proposed research is to combine a new computational approach with complementary NMR methods for characterizing binding of small ligands to proteins. This will address a wide need for defining interactions of proteins with natural ligands, such as peptides, non-peptidic biomolecules, or with non-natural ligands, including drug leads or other chemicals. While this Component 2 is on interactions of small ligands with proteins, this approach can readily be applied to protein-protein or protein-nucleic acid interactions. This connects this research to Components 1 and 4. Thus, both aspects address the problem of characterizing weak but specific iinteractions of proteins with small molecules and other macromolecules in situations where experimental constraints are sparse. We proposeto develop an approach that will build on the recently developed TreeDock algorithm and will combine it with information from NMR spectroscopy on small-molecule inhibitors of protein-protein interactions obtained from high-throughput library screening. To define structures of weak protein-inhibitor complexes, TreeDock can search the docking space very fast by eliminating up front all configurations in which the components to be docked have no contact or penetrate each other. This is achieved with kd search trees. The algorithm uses complete enumeration of the search space at high resolution and can handle ligand mobility by querying a database of docked rigid subgroups. TreeDock can be ideally interfaced with sparse data from NMR experiments. We propose NMR methods that can provide few specific protein-ligand contacts even under difficult experimental conditions, such as intermediate exchange or poor solubility. The combination of the computational and experimental NMR methods will also be capable of dealing with structural changes of receptor proteins upon ligand binding. The research will be pursued four Specific Aims: 1: Development of a flexible docking algorithm (TreeDock II) that is capable of screening compound libraries efficiently, docking peptides and DNA/RNA to proteins. 2: Development of a compiler (parser, classifier) of chemical compound libraries. 3. Experimental methods for measuring NOEs in the intermediate exchange regime and defining anchors for TreeDock - Application to inhibitors of Bcl-2 type proteins. 4. Define complexes of small-molecule inhibitors with translation initiation factors 4E and 4A. Harvard Medical School, Boston, MA PHS 398 (Rev. 04/06) Page 126 Form Page 2 Principal Investigator/Program Director (Last, First, Middle): Wagner, Gerhard - Component 2 KEY PERSONNEL. See instructions. Use continuationpages as needed to provide the required information in the format shown below. Start with Principal Investigator(s). List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Wagner, Gerhard GWAGNER Harvard Medical School PI Fahmy, Amr AMRFAHMY Harvard Medical School coPI OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Human Embryonic Stem Cells E] No CH Yes If the proposed project Involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://stemcells.nih.qov/registrv/index.asp. Use continuation pages as needed. If a specific line cannot be referencedat this time, include a statement that one from the Registry will be used. Cell Line PHS 398(Rev. 04/06) Page 127 Form Pa9e 2-continued Number the following pages consecutively throughout the application: Do not use suffixes such as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): Wagner, Gerhard - Component 2 DETAILED BUDGET FOR INITIAL BUDGET PERIOD DIRECT COSTS ONLY PERSONNEL (Applicant organization only) Months Devoted to Project ROLE ON Cal. Acad. Sum. INST.BASE NAME PROJECT Mnths Mnths Mnths SALARY Principal Gerhard Wagner 1.2 183,500 Investigator Co- Amr Fahmy 6 92,920 Investigator Research Mikhail Reibarkh 12 48,542 Fellow SUBTOTALS hwi CONSULTANT COSTS EQUIPMENT (Itemize) FROM THROUGH 5/1/07 4/30/08 DOLLAR AMOUNT REQUESTED (omit cents) SALARY FRINGE REQUESTED BENEFITS TOTAL 18,350 4,597 22,947 46,460 11,638 58,098 48,542 11,626 60,168 113,352 27,861 141,2131 . SUPPLIES (Itemize by category) NMR tubes, Shigemi tubes 1,000 Labeled amino acids 2,000 Chemicals and disposables 2,000 Precursors for ILV,LV or L labeling 4,000 Enzymes for cloning 3,000 15NH3 , 13C glucose &D2O 1 5,000 PCR primers 3,000 30,000 TRAVEL Costs for 2 researchers to attend a relevant conference 3,000 PATIENT CARE COSTS INPATIENT - OUTPATIENT - ALTERATIONS AND RENOVATIONS (Itemize by category) _ OTHER EXPENSES (Itemize by category) NMR Spectrometer maintenance, repair &operating costs (detailed in Justification) $23,000 23,000 CONSORTIUM/CONTRACTUAL COSTS DIRECT COSTS SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD (Item 7a, Face Page) $ 197,213 CONSORTIUM/CONTRACTUAL COSTS FACILITIES AND ADMINISTRATIVE COSTS TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD $ 197,213 PHS 398 (Rev. 04/06) Page 128 Form Page 4